Synthetic antigens for the detection of antibodies to hepatitis C virus

ABSTRACT

Peptide sequences are provided which are capable of mimicking proteins encoded by HCV for use as reagents for screening of blood and blood products for prior exposure to HCV. The peptides are at least 5 amino acids long and can be used in various specific assays for the detection of antibodies to HCV, for the detection of HCV antigens, or as immunogens.

This is a Divisional of application Ser. No. 08/391,671, filed Feb. 21,1995, which is a Continuation of Ser. No. 07/920,286, filed Oct. 14,1992 abandoned, which is a continuation of PCT/EP91/024 filed Dec. 13,1991.

The implementation of systematic testing for hepatitis B virus (HBV) hasbeen instrumental in eliminating this virus from the blood supply.Nevertheless, a significant number of post-transfusion hepatitis (PTH)cases still occur. These cases are generally attributable to non-A,non-B hepatitis (NANBH) virus(es), the diagnosis of which is usuallymade by exclusion of other viral markers.

The etiological agent responsible for a large proportion of these caseshas recently been cloned (Choo, Q-L et al. Science (1988) 244:359-362)and a first-generation antibody test developed (Kuo, G. et al. Science(1989) 244:362-364). The agent has been identified as apositive-stranded RNA virus, and the sequence of its genome has beenpartially determined. Studies suggest that this virus, referred tosubsequently as hepatitis C virus (HCV), may be related to flavivirusesand pestiviruses. A portion of the genome of an HCV isolated from achimpanzee (HCV_(CDC/CHI)) is disclosed in EPO 883109225. The codingsequences disclosed in this document do not include sequencesoriginating from the 5'-end of the viral genome which code for putativestructural proteins. Recently however, sequences derived from thisregion of the HCV genome have been published (Okamoto, H. et al., JapanJ. Exp. Med. 60:167-177, 1990.). The amino acid sequences encoded by theJapanese clone HC-J1 were combined with the HCV_(CDC/CHI) sequences in aregion where the two sequences overlap to generate the compositesequence depicted in FIG. 1. Specifically, the two sequences were joinedat glycine₄₅₁. It should be emphasized that the numbering system usedfor the HCV amino acid sequence is not intended to be absolute since theexistence of variant HCV strains harboring deletions or insertions ishighly probable. Sequences corresponding to the 5' end of the HCV genomehave also recently been disclosed in EPO 90302866.0.

In order to detect potential carriers of HCV, it is necessary to haveaccess to large amounts of viral proteins. In the case of HCV, there iscurrently no known method for culturing the virus, which precludes theuse of virus-infected cultures as a source of viral antigens. Thecurrent first-generation antibody test makes use of a fusion proteincontaining a sequence of 363 amino acids encoded by the HCV genome. Itwas found that antibodies to this protein could be detected in 75 to 85%of chronic NANBH patients. In contrast, only approximately 15% of thosepatients who were in the acute phase of the disease, had antibodieswhich recognized this fusion protein (Kuo, G. et al. Science (1989)244:362-364). The absence of suitable confirmatory tests, however, makesit difficult to verify these statistics. The seeming similarity betweenthe HCV genome and that of flaviviruses makes it possible to predict thelocation of epitopes which are likely to be of diagnostic value. Ananalysis of the HCV genome reveals the presence of a continuous longopen reading frame. Viral RNA is presumably translated into a longpolyprotein which is subsequently cleaved by cellular and/or viralproteases. By analogy with, for example, Dengue virus, the viralstructural proteins are presumed to be derived from the amino-terminalthird of the viral polyprotein. At the present time, the precise sitesat which the polyprotein is cleaved can only be surmised. Nevertheless,the structural proteins are likely to contain epitopes which would beuseful for diagnostic purposes, both for the detection of antibodies aswell as for raising antibodies which could subsequently be used for thedetection of viral antigens. Similarly, domains of nonstructuralproteins are also expected to contain epitopes of diagnostic value, eventhough these proteins are not found as structural components of virusparticles.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D show the amino acid sequence of the compositeHCV_(HC-J1/CDC/CHI) (SEQ ID NO:23)

FIGS. 2A-2L show the antibody binding to individual peptides and variousmixtures in an ELISA assay. Coating combinations used for FIGS. 2A-2Lare 1:IX, 2:XVIII, 3:I, 4:III, 5:V, 6:IX+XVIII, 7:I+XVIII, 8:I+III+IX,9:I+III+V+XVIII, 10:I+III+V+IX, 11:I+III+IX+XVIII, 12:I+III+V+IX+XVIII.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

It is known that RNA viruses frequently exhibit a high rate ofspontaneous mutation and, as such, it is to be expected that no two HCVisolates will be completely identical, even when derived from the sameindividual. For the purpose of this disclosure, a virus is considered tobe the same or equivalent to HCV if it exhibits a global homology of 60percent or more with the HCV_(HC-J1/CDC/CHI) composite sequence at thenucleic acid level and 70 percent at the amino acid level.

Peptides are described which immunologically mimic proteins encoded byHCV. In order to accommodate strain-to-strain variations in sequence,conservative as well as non-conservative amino acid substitutions may bemade. These will generally account for less than 35 percent of aspecific sequence. It may be desirable in cases where a peptidecorresponds to a region in the HCV polypeptide which is highlypolymorphic, to vary one or more of the amino acids so as to bettermimic the different epitopes of different viral strains.

The peptides of interest will include at least five, sometimes six,sometimes eight, sometimes twelve, usually fewer than about fifty, moreusually fewer than about thirty-five, and preferably fewer than abouttwenty-five amino acids included within the sequence encoded by the HCVgenome. In each instance, the peptide will preferably be as small aspossible while still maintaining substantially all of the sensitivity ofthe larger peptide. It may also be desirable in certain instances tojoin two or more peptides together in one peptide structure.

It should be understood that the peptides described need not beidentical to any particular HCV sequence, so long as the subjectcompounds are capable of providing for immunological competition with atleast one strain of HCV. The peptides may therefore be subject toinsertions, deletions, and conservative or non-conservative amino acidsubstitutions where such changes might provide for certain advantages intheir use.

Substitutions which are considered conservative are those in which thechemical nature of the substitute is similar to that of the originalamino acid. Combinations of amino acids which could be consideredconservative are Gly, Ala; Asp, Glu; Asn, Gln; Val, Ile, Leu; Ser, Thr;Lys, Arg; and Phe, Tyr.

Furthermore, additional amino acids or chemical groups may be added tothe amino- or carboxyl terminus for the purpose of creating a "linkerarm" by which the peptide can conveniently be attached to a carrier. Thelinker arm will be at least one amino acid and may be as many as 60amino acids but will most frequently be 1 to 10 amino acids. The natureof the attachment to a solid phase or carrier need not be covalent.

Natural amino acids such as cysteine, lysine, tyrosine, glutamic acid,or aspartic acid may be added to either the amino- or carboxyl terminusto provide functional groups for coupling to a solid phase or a carrier.However, other chemical groups such as, for example, biotin andthioglycolic acid, may be added to the termini which will endow thepeptides with desired chemical or physical properties. The termini ofthe peptides may also be modified, for example, by N-terminalacetylation or terminal carboxy-amidation. The peptides of interest aredescribed in relation to the composite amino acid sequence shown inFIG. 1. The amino acid sequences are given in the conventional anduniversally accepted three-letter code. In addition to the amino acidsshown, other groups are defined as follows: Y is, for example, NH₂, oneor more N-terminal amino acids, or other moieties added to facilitatecoupling. Y may itself be modified by, for example, acetylation. Z is abond, (an) amino acid(s), or (a) chemical group(s) which may be used forlinking. X is intended to represent OH, NH₂, or a linkage involvingeither of these two groups.

Peptide I, shown in SEQ ID NO:1, corresponds to amino acids 1 to 20 andhas the amino acid sequence:

    Y-Met-Ser-Thr-Ile-Pro-Lys-Pro-Gln-Arg-Lys-Thr-Lys-Arg-Asn-Thr-Asn-Arg-Arg-    Pro-Gln-Z-X.                                         (I)                  

Peptide II, shown in SEQ ID NO:2, corresponds to amino acids 7 to 26 andhas the amino acid sequence:

    Y-Pro-Gln-Arg-Lys-Thr-Lys-Arg-Asn-Thr-Asn-Arg-Arg-Pro-Gln-Asp-Val-Lys-Phe-    Pro-Gly-Z-X.                                         (II)                 

Of particular interest is the oligopeptide IIA, shown in SEQ ID NO:3,which has the sequence:

IIA

Y-Gln-Arg-Lys-Thr-Lys-Arg-Asn-Thr-Asn-Arg-Arg-Z-X

Peptide III, shown in SEQ ID NO:4 corresponds to amino adds 13 to 32 andhas the sequence:

    Y-Arg-Asn-Thr-Asn-Arg-Arg-Pro-Gln-Asp-Val-Lys-Phe-Pro-Gly-Gly-Gly-Gln-Ile-    Val-Gly-Z-X.                                         (III)                

Peptide IV, shown in SEQ ID NO:5, corresponds to amino acid 37 to 56 andhas the sequence:

    Y-Leu-Pro-Arg-Arg-Gly-Pro-Arg-Leu-Gly-Val-Arg-Ala-Thr-Arg-Lys-Thr-Ser-Glu-    Arg-Ser-Z-X.                                         (IV)                 

Peptide V, shown in SEQ ID NO:6, corresponds to amino acids 49 to 68 andhas the sequence:

    Y-Thr-Arg-Lys-Thr-Ser-Glu-Arg-Ser-Gln-Pro-Arg-Gly-Arg-Arg-Gln-Pro-Ile-Pro-    Lys-Val-Z-X.                                         (V)                  

Peptide VI, shown in SEQ ID No:7, corresponds to amino acid 61 to 80 andhas the following sequence:

    Y-Arg-Arg-Gln-Pro-Ile-Pro-Lys-Val-Arg-Arg-Pro-Glu-Gly-Arg-Thr-Trp-Ala-Gln-    Pro-Gly-Z-X.                                         (VI)                 

Peptide VII, shown in SEQ ID NO:8, corresponds to amino acids 73 to 92and has the sequence:

    Y-Gly-Arg-Thr-Trp-Ala-Gln-Pro-Gly-Tyr-Pro-Trp-Pro-Leu-Tyr-Gly-Asn-Glu-Gly-    Cys-Gly-Z-X.                                         (VII)                

Peptide VIII, shown in SEQ ID NO:9, corresponds to amino acids 1688 to1707 and has the sequence:

    Y-Leu-Ser-Gly-Lys-Pro-Ala-Ile-Ile-Pro-Asp-Arg-Glu-Val-Leu-Tyr-Arg-Glu-Phe-    Asp-Glu-Z-X.                                         (VIII)               

Peptide IX, shown in SEQ ID NO:10, corresponds to amino acids 1694 to1713 and has the sequence:

    Y-Ile-Ile-Pro-Asp-Arg-Glu-Val-Leu-Tyr-Arg-Glu-Phe-Asp-Glu-Met-Glu-Glu-Cys-    Ser-Gln-Z-X.                                         (IX)                 

Peptide X, shown in SEQ ID No:11, corresponds to amino acids 1706 to1725 and has the sequence:

    Y-Asp-Glu-Met-Glu-Glu-Cys-Ser-Gln-His-Leu-Pro-Tyr-Ile-Glu-Gln-Gly-Met-Met-    Leu-Ala-Z-X.                                         (X)                  

Peptide XI, shown in SEQ ID No:12, corresponds to amino acids 1712 to1731 and has the sequence:

    Y-Ser-Gln-His-Leu-Pro-Tyr-Ile-Glu-Gln-Gly-Met-Met-Leu-Ala-Glu-Gln-Phe-Lys-    Gln-Lys-Z-X.                                         (XI)                 

Peptide XII, shown in SEQ ID No:13, corresponds to amino acids 1718 to1737 and has the sequence:

    Y-Ile-Glu-Gln-Gly-Met-Met-Leu-Ala-Glu-Gln-Phe-Lys-Gln-Lys-Ala-Leu-Gly-Leu-    Leu-Gln-Z-X.                                         (XII)                

Peptide XIII, shown in SEQ ID NO:14, corresponds to amino acids 1724 to1743 and has the sequence:

    Y-Leu-Ala-Glu-Gln-Phe-Lys-Gln-Lys-Ala-Leu-Gly-Leu-Leu-Gln-Thr-Ala-Ser-Arg-    Gln-Ala-Z-X.                                         (XIII)               

Peptide XIV, shown in SEQ ID NO:15, corresponds to amino acids 1730 to1749 and has the sequence:

    Y-Gln-Lys-Ala-Leu-Gly-Leu-Leu-Gln-Thr-Ala-Ser-Arg-Gln-Ala-Glu-Val-Ile-Ala-    Pro-Ala-Z-X.                                         (XIV)                

Peptide XV, shown in SEQ ID No:16, corresponds to amino acids 2263 to2282 and has the sequence:

    Y-Glu-Asp-Glu-Arg-Glu-Ile-Ser-Val-Pro-Ala-Glu-Ile-Leu-Arg-Lys-Ser-Arg-Arg-    Phe-Ala-Z-X.                                         (XV)                 

Peptide XVI, shown in SEQ ID NO:17, corresponds to amino acids 2275 to2294 and has the sequence:

    Y-Leu-Arg-Lys-Ser-Arg-Arg-Phe-Ala-Gln-Ala-Leu-Pro-Val-Trp-Ala-Arg-Pro-Asp-    Tyr-Asn-Z-X.                                         (XVI)                

Peptide XVII, shown in SEQ ID NO:18, corresponds to amino acids 2287 to2306 and has the sequence:

    Y-Val-Trp-Ala-Arg-Pro-Asp-Tyr-Asn-Pro-Pro-Leu-Val-Glu-Thr-Trp-Lys-Lys-Pro-    Asp-Tyr-Z-X.                                         (XVII)               

Peptide XVIII, shown in SEQ ID NO:19, corresponds to amino acids 2299 to2318 and has the sequence:

    Y-Glu-Thr-Trp-Lys-Lys-Pro-Asp-Tyr-Glu-Pro-Pro-Val-Val-His-Gly-Cys-Pro-Leu-    Pro-Pro-Z-X.                                         (XVIII)              

Peptide XIX, shown in SEQ ID NO:20, corresponds to amino acids 2311 to2330 and has the sequence:

    Y-Val-His-Gly-Cys-Pro-Leu-Pro-Pro-Pro-Lys-Ser-Pro-Pro-Val-Pro-Pro-Pro-Arg-    Lys-Lys-Z-X.                                         (XIX)                

Of particular interest is the use of the mercapto-group of cysteines orthioglycolic acids used for acylating terminal amino groups forcyclizing the peptides or coupling two peptides together. Thecyclization or coupling may occur via a single bond or may beaccomplished using thiol-specific reagents to form a molecular bridge.

The peptides may be coupled to a soluble carrier for the purpose ofeither raising antibodies or facilitating the adsorption of the peptidesto a solid phase. The nature of the carrier should be such that it has amolecular weight greater than 5000 and should not be recognized byantibodies in human serum. Generally, the crier will be a protein.Proteins which are frequently used as carriers are keyhole limpethemocyanin, bovine gamma globulin, bovine serum albumin, andpoly-L-lysine.

There are many well described techniques for coupling peptides tocarriers. The linkage may occur at the N-terminus, C-terminus or at aninternal site in the peptide. The peptide may also be derivatized forcoupling. Detailed descriptions of a wide variety of coupling proceduresare given, for example, in Van Regenmortel M. H. V., Briand, J. P.,Muller, S., and Plaue, S., Laboratory Techniques in Biochemistry andMolecular Biology, Vol. 19, Synthetic Polypeptides as Antigens, ElsevierPress, Amsterdam, N.Y., Oxford, 1988.

The peptides may also be synthesized directly on an oligo-lysine core inwhich both the alpha as well as the epsilon-amino groups of lysines areused as growth points for the peptides. The number of lysines comprisingthe core is preferably 3 or 7. Additionally, a cysteine may be includednear or at the C-terminus of the complex to facilitate the formation ofhomo- or heterodimers. The use of this technique has been amplyillustrated for hepatitis B antigens (Tam, J. P., and Lu, Y-A., Proc.Natl. Acad. Sci. USA (1989) 86:9084-9088) as well as for a variety ofother antigens (see Tam, J. P., Multiple Antigen Peptide System: A NovelDesign for Synthetic Peptide Vaccine and Immunoassay, in SyntheticPeptides, Approaches to Biological Problems, Tam, J. P., and Kaiser, E.T., ed. Alan R. Liss Inc., New York, 1989).

Depending on their intended use, the peptides may be either labeled orunlabeled. Labels which may be employed may be of any type, such asenzymatic, chemical, fluorescent, luminescent, or radioactive. Inaddition, the peptides may be modified for binding to surfaces or solidphases, such as, for example, microtiter plates, nylon membranes, glassor plastic beads, and chromatographic supports such as cellulose,silica, or agarose. The methods by which peptides can be attached orbound to solid support or surface are well known to those versed in theart.

Of particular interest is the use of mixtures of peptides for thedetection of antibodies specific for hepatitis C virus. Mixtures ofpeptides which are considered particularly advantageous are:

A. II, III, V, IX, and XVIII

B. I, II, V, IX, XI, XVI, and XVIII

C. II, III, IV, V, VIII, XI, XVI, and XVIII

D. II, IX, and XVIII

E. II, III, IV, and V

F. VIII, IX, XI, XIII, and XIV

G. XV, XVI, XVII, XVIII, and XIX

Antibodies which recognize the peptides can be detected in a variety ofways. A preferred method of detection is the enzyme-linked immunosorbantassay (ELISA) in which a peptide or mixture of peptides is bound to asolid support. In most cases, this will be a microtiter plate but may inprinciple be any sort of insoluble solid phase. A suitable dilution ordilutions of serum or other body fluid to be tested is brought intocontact with the solid phase to which the peptide is bound. Theincubation is carried out for a tie necessary to allow the bindingreaction to occur. Subsequently, unbound components are removed bywashing the solid phase. The detection of immune complexes is achievedusing antibodies which specifically bind to human immunoglobulins, andwhich have been labeled with an enzyme, preferably but not limited toeither horseradish peroxidase, alkaline phosphatase, orbeta-galactosidase, which is capable of converting a colorless or nearlycolorless substrate or co-substrate into a highly colored product or aproduct capable of forming a colored complex with a chromogen.Alternatively, the detection system may employ an enzyme which, in thepresence of the proper substrate(s), emits light. The amount of productformed is detected either visually, spectrophotometrically,electrochemically, or luminometrically, and is compared to a similarlytreated control. The detection system may also employ radioactivelylabeled antibodies, in which case the amount of immune complex isquantified by scintillation counting or gamma counting.

Other detection systems which may be used include those based on the useof protein A derived from Staphylococcus aureus Cowan strain I, proteinG from group C Staphylococcus sp. (strain 26RP66), or systems which makeuse of the high affinity biotin-avidin or streptavidin binding reaction.

Antibodies raised to carrier-bound peptides can also be used inconjunction with labeled peptides for the detection of antibodiespresent in serum or other body fluids by competition assay. In thiscase, antibodies raised to carrier-bound peptides are attached to asolid support which may be, for example, a plastic bead or a plastictube. Labeled peptide is then mixed with suitable dilutions of the fluidto be tested and this mixture is subsequently brought into contact withthe antibody bound to the solid support. After a suitable incubationperiod, the solid support is washed and the amount of labeled peptide isquantified. A reduction in the amount of label bound to the solidsupport is indicative of the presence of antibodies in the originalsample. By the same token, the peptide may also be bound to the solidsupport. Labeled antibody may then be allowed to compete with antibodypresent in the sample under conditions in which the amount of peptide islimiting. As in the previous example, a reduction in the measured signalis indicative of the presence of antibodies in the sample tested.

Another preferred method of antibody detection is the homogeneousimmunoassay. There are many possible variations in the design of suchassays. By way of example, numerous possible configurations forhomogeneous enzyme immunoassays and methods by which they may beperformed are given in Tijssen, P., Practice and Theory of EnzymeImmunoassays, Elsevier Press, Amersham, Oxford, N.Y. 1985. Detectionsystems which may be employed include those based on enzyme channeling,bioluminescence, allosteric activation and allosteric inhibition.Methods employing liposome-entrapped enzymes or coenzymes may also beused (see Pinnaduwage, P. and Huang, L, Clin. Chem. (1988) 34/2:268-272, and Ullman, E. F. et al., Clin. Chem. (1987) 33/9: 1579-1584for examples).

The synthesis of the peptides can be achieved in solution or on a solidsupport. Synthesis protocols generally employ the uset-butyloxycarbonyl- or 9-fluorenylmethoxy-carbonyl-protected activatedamino acids. The procedures for carrying out the syntheses, the types ofside-chain protection, and the cleavage methods are amply described in,for example, Stewart and Young, Solid Phase Peptide Synthesis, 2ndEdition, Pierce Chemical Company, 1984; and Atherton and Sheppard, SolidPhase Peptide Synthesis, IRL Press, 1989.

EXPERIMENTAL I. Peptide Synthesis

All of the peptides described were synthesized on Pepsyn Kpolyamide-Kieselguhr resin (Milligen, Novato, Calif.) which had beenfunctionalized with ethylenediamine and onto which the acid-labilelinker 4-(alpha-Fmoc-amino-2',4'-dimethoxybenzyl) phenoxyacetic acid hadbeen coupled (Rink, Tetrahedron Lett. (1987) 28:3787). t-Butyl-basedside-chain protection and Fmoc alpha-amino-protection was used. Theguanidino-group of arginine was protected by the2,2,5,7,8-pentamethylchroman-6-sulfonyl moiety. The imidazole group ofhistidine was protected by either t-Boc or trityl and the sulfhydrylgroup of cysteine was protected by a trityl group. Couplings werecarried out using performed O-pentafluorophenyl esters except in thecase of arginine where diisopropylcarbodiimide-mediatedhydroxybenzotriazole ester formation was employed. Except for peptide Lall peptides were N-acetylated using acetic anhydride. All syntheseswere carried out on a Milligen 9050 PepSynthesizer (Novato, Calif.)using continuous flow procedures. Following cleavage withtrifluoroacetic acid in the presence of scavengers and extraction withdiethylether, all peptides were analyzed by C₁₈ -reverse phasechromatography.

II. Detection of Antibodies to Hepatitis C Virus

A. Use of Peptides Bound to a Nylon Membrane

Peptides were dissolved in a suitable buffer to make a concentratedstock solution which was then further diluted in phosphate-bufferedsaline (PBS) or sodium carbonate buffer, pH 9.6 to make workingsolutions. The peptides were applied as lines on a nylon membrane (Pall,Portsmouth, United Kingdom), after which the membrane was treated withcasein to block unoccupied binding sites. The membrane was subsequentlycut into strips perpendicular to the direction of the peptide lines.Each strip was then incubated with a serum sample diluted 1 to 100,obtained from an HCV-infected individual. Antibody binding was detectedby incubating the strips with goat anti-human immunoglobulin antibodiesconjugated to the enzyme alkaline phosphatase. After removing unboundconjugate by washing, a substrate solution containing5-bromo-4-chloro-3-indolylphosphate and nitro blue tetrazolium wasadded.

Positive reactions are visible as colored lines corresponding to thepositions of the peptides which are specifically recognized. Thereaction patterns of thirty-six different sera are tabulated in Table 1.The results shown in Table 1 are further summarized in Table 2.

B. Use of Peptides in an Enzyme-Linked Immunosorbent Assay (ELISA)

Peptide stock solutions were diluted in sodium carbonate buffer, pH 9.6and used to coat microtiter plates at a peptide concentration of 2micrograms per milliliter. A mixture consisting of peptides II, III, V,IX, and XVIII was also used to coat plates. Following coating, theplates were blocked with casein. Fifteen HCV-antibody-positive sera andcontrol sera from seven uninfected blood donors were diluted 1 to 20 andincubated in wells of the peptide-coated plates. Antibody binding wasdetected by incubating the plates with goat anti-human immunoglobulinantibodies conjugated to the enzyme horseradish peroxidase. Followingremoval of unbound conjugate by washing, a solution containing H₂ O₂ and3,3',5,5'-tetramethylbenzidine was added. Reactions were stopped after asuitable interval by addition of sulfuric acid. Positive reactions gaverise to a yellow color which was quantified using a conventionalmicrotiter plate reader. The results of these determinations aretabulated in Table 3. To correct for any aspecific binding which couldbe attributable to the physical or chemical properties of the peptidesthemselves, a cut-off value was determined for each peptideindividually. This cut-off absorbance value was calculated as theaverage optical density of the negative samples plus 0.200. Samplesgiving absorbance values higher than the cut-off values are consideredpositive. The results for the fifteen positive serum samples are furthersummarized in Table 4.

While it is evident that some of the peptides are recognized by a largepercentage of sera from HCV-infected individuals, it is also clear thatno single peptide is recognized by all sera. In contrast, the peptidemixture was recognized by all fifteen sera and, for six of the fifteensera, the optical densities obtained were equal to or higher than thoseobtained for any of the peptides individually. These results serve toillustrate the advantages of using mixtures of peptides for thedetection of anti-HCV antibodies.

C. Binding of Antibodies in Sera from HCV-Infected Patients to VariousIndividual Peptides and Peptide Mixtures in an ELISA

Five peptides were used individually and in seven different combinationsto coat microtiter plates. The plates were subsequently incubated withdilutions of fifteen HCV antibody-positive sera in order to evaluate therelative merits of using mixtures as compared to individual peptides forantibody detection. The mixtures used and the results obtained are shownin FIG. 2.

In general, the mixtures functioned better than individual peptides.This was particularly evident for mixture 12 (peptides I, III, V, IX,and XVIII) which was recognized by all twelve of the sera tested. Theseresults underscore the advantages of using mixtures of peptides indiagnostic tests for the detection of antibodies to HCV.

D. Use of a Mixture of Peptides in an ELISA Assay for the Detection ofAnti-HCV Antibodies

A mixture of peptides II, III, V, IX, and XVIII was prepared and used tocoat microtiter plates according to the same procedure used to test theindividual peptides. A total of forty-nine sera were tested frompatients with clinically diagnosed but undifferentiated chronic non Anon B hepatitis as well as forty-nine sera from healthy blood donors.Detection of antibody binding was accomplished using goat anti-humanimmunoglobulin antibodies conjugated to horseradish peroxidase. Theresulting optical density values are given in Table 5. These resultsindicate that the mixture of peptides is not recognized by antibodies insera from healthy donors (0/49 reactives) but is recognized by a largeproportion. (41/49, or 84%) of the sera from patients with chronicNANBH. These results demonstrate that the peptides described can be usedeffectively as mixtures for the diagnosis of HCV infection.

E. Detection of Anti-HCV Antibodies in Sera from Patients with AcuteNANB Infection using Individual Peptides Bound to Nylon Membranes and aMixture of Peptides in an ELISA Assay, and Comparison with aCommercially Available Kit

Peptides were applied to nylon membranes or mixed and used to coatmicrotiter plates as previously described. The peptide mixture consistedof peptides II, III, V, IX, and XVIII. Sera obtained from twenty-ninepatients with acute non-A, non-B hepatitis were then tested for thepresence of antibodies to hepatitis C virus. These same- sera were alsoevaluated using a commercially available kit (Ortho, Emeryville, Calif.,USA).

The results of this comparative study are given in Table 6. In order tobe able to compare the peptide-based ELISA with the commerciallyavailable kit, the results for both tests are also expressed as signalto noise ratios (S/N) which were calculated by dividing the measuredoptical density obtained for each sample by the cut-off value. Asignal-to-noise ratio greater or equal to 1.0 is taken to represent apositive reaction. For the commercially available kit, the cut-off valuewas calculated according to the manufacturer's instructions. The cut-offvalue for the peptide-based ELISA was calculated as the average opticaldensity of five negative samples plus 0.200.

The scale used to evaluate antibody recognition of nylon-bound peptideswas the same as that given in Table 1. Of the twenty-nine samplestested, twenty-five (86%) were positive in the peptide-based ELISA andrecognized one or more nylon-bound peptides. In contrast, only fourteenof the twenty-nine sera scored positive in the commercially availableELISA. These results serve to illustrate the advantages of using peptidemixtures for the detection of anti-HIV antibodies as well as the need toinclude in the mixtures peptides which contain amino acid sequencesderived from different regions of the HCV polyprotein.

                  TABLE 1                                                         ______________________________________                                        Recognition of peptides bound to nylon membranes by                             sera from persons infected by HCV.                                                 PEPTIDE                                                                Serum nr.                                                                            I      II     III  IV   V    VI   VII  VIII IX                         ______________________________________                                           1     3 1       1                                                             2                                                                             3 1 0.5 2 1  0.5  2 0.5                                                       4                                                                             6   2 1 0.5                                                                   7 0.5 1 2 1 0.5   3 2                                                         8 0.5 1 3 1 1  1 1                                                           10  1 0.5     3 1                                                             13 0.5 0.5 2  0.5                                                             15    0.5    2 1                                                              16 2 1 0.5 0.5 1 0.5  2 0.5                                                   18 1 1 3 0.5  2 0.5                                                           23 0.5  1 1   0.5                                                             24 1 0.5 2 1 0.5 0.5 0.5 2                                                    25   1 0.5    2 0.5                                                           26        1                                                                   27 0.5 0.5 1     3 2                                                          29  0.5 3 2 1 1 0.5                                                           30  0.5 0.5 1 1 0.5                                                           31  1 0.5                                                                     32  1 2                                                                       33        0.5                                                                 34  1 1 1    3 1                                                              35 1 1 2 1 1 1 0.5                                                            36 1  2 1  1                                                                  37 1 1                                                                        44  1 2 1 0.5                                                                 46  0.5 2 0.5 0.5  0.5 2                                                      47  0.5  0.5  0.5                                                             48 1 2  2   0.5 2                                                             49  1 1 0.5 0.5 0.5                                                           50  1 2 1    2 0.5                                                            51   2 0.5 0.5  0.5                                                           52   2 0.5   0.5                                                              54   2  0.5 0.5 1 0.5                                                         56 ND ND ND ND ND ND ND 2                                                   ______________________________________                                        Se-                                                                             rum PEPTIDE                                                                       nr.    X   XI  XII XIII XIV  XV   XVI  XVII XVIII                                                   XIX                                               ______________________________________                                           1   0.5         2 2 1 1                                                       2  0.5  1 2  2 1                                                              3 1 0.5                                                                       4    1                                                                        6       2                                                                     7 2 1  1  2  0.5 1 1                                                          8 2 1  1   1 1 0.5                                                           10 1    0.5 2 2  2 2                                                          13 1   1 0.5  0.5                                                             15 0.5    1  0.5                                                              16 1     2 2 1 2                                                              18       1 0.5                                                                23 1 0.5  0.5  0.5                                                            24       1                                                                    25 0.5 2  1 1  2                                                              26       0.5                                                                  27       1 2 1 0.5                                                            29 2 1  1 1 2 2 1 1 1                                                         30                                                                            31 0.5 0.5    0.5                                                             32 1 0.5    1 1                                                               33 1 0.5       0.5                                                            34       1 1 0.5                                                              35                                                                            36                                                                            37                                                                            44      2  0.5                                                                46                                                                            47         1 1                                                                48    0.5  1 1  1 0.5                                                         49 0.5 0.5  0.5    1                                                          50 1 1  1   1 1 0.5 0.5                                                       51       1 1 0.5                                                              52 0.5                                                                        54 1 1  1   1 1 1                                                             56  0.5 1 2 1                                                               ______________________________________                                         Blank: no reaction; 0.5: weakly positive; 1: clearly positive; 2: strong      reaction; 3: intense reaction; ND: not determined                        

                  TABLE 2                                                         ______________________________________                                        Summary of antibody binding to nylon-bound HCV peptides                         by sera from infected patients.                                                  Peptide No. reactive sera % reactive sera                                ______________________________________                                        I        13/35             37                                                   II 22/35 63                                                                   III 27/35 77                                                                  IV 24/35 69                                                                   V 14/35 40                                                                    VI 11/35 31                                                                   VII 11/35 31                                                                  VIII 19/36 53                                                                 IX 9/36 25                                                                    X 17/36 47                                                                    XI 15/36 42                                                                   XII 1/36 3                                                                    XIII 13/36 36                                                                 XIV 7/36 19                                                                   XV 9/36 25                                                                    XVI 20/36 56                                                                  XVII 14/36 39                                                                 XVIII 14/36 39                                                                XIX 8/36 22                                                                 ______________________________________                                    

                                      TABLE 3                                     __________________________________________________________________________    Comparison of Individual Peptides in an ELISA Assay                             for the Detection of Antibodies to HCV.                                     sample                                                                             peptide                                                                  indent                                                                             I   II  III                                                                              IV  V   VI  VII                                                                              VIII                                                                              IX                                         __________________________________________________________________________       1 0.786 1.119 1.284 0.265 0.042 0.04 0.05 0.571 0.659                         2 0.044 0.039 0.11 0.041 0.037 0.038 0.039 0.479 0.78                         3 0.815 0.944 0.825 0.399 0.654 0.487 0.32 0.705 0.965                        7 1.122 1.23 0.588 0.682 0.659 0.182 0.107 0.907 1.42                         8 1.155 1.159 1.2 0.508 1.272 0.433 0.623 0.61 0.863                         10 1.089 1.236 1.083 0.044 0.508 0.042 0.073 1.49 1.529                       11 0.048 0.051 0.476 0.052 0.119 0.039 0.1 0.634 0.711                        15 0.224 0.602 0.813 0.093 0.068 0.077 0.147 0.807 1.225                      23 0.62 0.8 0.924 0.568 0.759 0.442 0.683 0.089 0.121                         24 1.042 1.132 1.026 0.518 0.916 0.302 0.253 1.013 1.364                      49 0.624 0.73 0.884 0.171 0.372 0.055 0.04 0.084 0.064                        13 0.76 0.857 0.815 0.087 0.422 0.098 0.045 0.473 0.489                       31 0.84 1.114 0.445 0.672 0.046 0.041 0.042 0.184 0.15                        47 1.303 1.53 1.236 0.751 0.83 0.629 0.073 0.545 0.739                        56 1.169 1.301 1.364 1.269 1.374 0.85 1.066 1.45 1.523                        bd A28 0.054 0.043 0.139 0.045 0.135 0.042 0.041 0.086 0.115                  bd A169 0.041 0.042 0.134 0.044 0.038 0.04 0.41 0.061 0.07                    bd A170 0.04 0.044 0.117 0.04 0.036 0.04 0.04 0.061 0.05                      bd A171 0.041 0.046 0.148 0.043 0.037 0.045 0.045 0.077 0.065                 bd A166 0.047 0.046 0.124 0.044 0.038 0.042 0.041 0.056 0.066                 bd A165 0.041 0.046 0.123 0.043 0.035 0.051 0.042 0.051 0.091                 AVG 0.044 0.045 0.131 0.43 0.053 0.043 0.042 0.069 0.076                      STD 0.005 0.002 0.011 0.002 0.037 0.004 0.002 0.013 0.021                     cut off 0.109 0.101 0.214 0.099 0.214 0.105 0.098 0.158 0.189               __________________________________________________________________________    sample                                                                             peptide                                                                  indent                                                                             X  XI  XII                                                                              XIII                                                                             XIV XV XVI                                                                              XVII                                                                              XVIII                                                                             XIX                                       __________________________________________________________________________       1 0.048 0.64 0.043 0.068 0.044 0.041 1.063 0.956 1.383 1.346                  2 0.169 0.563 0.039 0.042 0.515 0.039 0.64 0.319 0.154 0.49                   3 0.468 0.668 0.041 0.093 0.341 0.043 0.292 0.038 0.046 0.038                 7 0.663 0.646 0.041 0.235 0.068 0.515 0.012 0.041 0.872 1.211                 8 0.752 1.175 0.046 0.42 0.102 0.068 0.552 0.671 0.417 0.058                 10 0.689 0.834 0.041 0.044 0.314 0.193 0.886 0.037 1.335 1.356                11 0.199 0.967 0.125 0.454 0.088 0.111 0.274 0.093 0.838 0.065                15 0.315 0.668 0.046 0.154 0.202 0.065 0.372 0.091 0.155 0.017                23 0.422 0.896 0.041 0.049 0.101 0.068 0.311 0.038 0.052 0.05                 24 0.236 0.397 0.054 0.123 0.076 0.051 0.418 0.053 0.1 0.085                  49 0.209 0.731 0.044 0.113 0.039 0.044 0.299 0.038 0.192 0.041                13 0.529 0.735 0.043 0.044 0.186 0.013 0.086 0.037 0.065 0.04                 31 0.255 0.69 0.041 0.04 0.061 0.136 0.292 0.038 0.224 0.501                  47 0.044 0.041 0.041 0.041 0.498 0.04 0.268 0.042 1.288 1.206                 56 0.079 1.069 0.058 0.568 0.038 0.039 0.218 0.036 0.087 0.039                bd A28 0.044 0.042 0.044 0.052 0.043 0.043 0.307 0.042 0.045 0.061                                               bd A169 0.043 0.042 0.041 0.04 0.041                                         0.041 0.255 0.038 0.056 0.042                                                  bd A170 0.04 0.039 0.04 0.035 0.035                                          0.144 0.292 0.036 0.058 0.039                                                  bd A171 0.043 0.041 0.043 0.039 0.04                                         0.045 0.286 0.037 0.05 0.04                 bd A166 0.041 0.011 0.042 0.04 0.041 0.041 0.207 0.039 0.046 0.041                                               bd A165 0.041 0.04 0.042 0.039 0.043                                         0.039 0.253 0.034 0.06 0.098                AVG 0.042 0.041 0.042 0.041 0.041 0.059 0.267 0.038 0.053 0.054                                                  STD 0.001 0.001 0.001 0.005 0.002                                            0.038 0.033 0.002 0.006 0.021                                                  cut off 0.095 0.094 0.095 0.106                                              0.097 0.223 0.416 0.084 0.121             __________________________________________________________________________                                        0.167                                 

                  TABLE 4                                                         ______________________________________                                        Summary of antibody-binding to individual peptides in an                        ELISA assay.                                                                     Peptide No. reactive sera % reactive sera                                ______________________________________                                        I        13                87                                                   II 13 87                                                                      III 14 93                                                                     IV 10 67                                                                      V 10 67                                                                       VI 7 47                                                                       VII 8 53                                                                      VIII 13 87                                                                    IX 12 80                                                                      X 13 87                                                                       XI 13 87                                                                      XII 1 7                                                                       XIII 7 47                                                                     XIV 8 53                                                                      XV 2 13                                                                       XVI 5 33                                                                      XVII 4 27                                                                     XVIII 10 67                                                                   XIX 6 40                                                                    ______________________________________                                    

                  TABLE 5                                                         ______________________________________                                        Use of a peptide mixture for the detection of antibodies to                     HCV in sera from chronic NANBH patients and comparison                        to sera from healthy blood donors                                               Chronic NANB Sera Control Sera                                            Serum nr. Optical Density                                                                           Serum nr. Optical Density                               ______________________________________                                        101       0.041       1         0.049                                           102 1.387 2 0.047                                                             103 1.578 3 0.049                                                             104 1.804 4 0.046                                                             105 1.393 5 0.049                                                             107 1.604 6 0.045                                                             108 1.148 7 0.043                                                             109 1.714 8 0.053                                                             110 1.692 9 0.049                                                             112 0.919 10 0.047                                                            113 1.454 11 0.060                                                            114 0.936 12 0.044                                                            115 0.041 13 0.049                                                            116 1.636 14 0.051                                                            118 1.242 15 0.056                                                            119 1.568 16 0.050                                                            120 1.290 17 0.049                                                            121 1.541 18 0.055                                                            122 1.422 19 0.054                                                            123 1.493 20 0.058                                                            124 1.666 21 0.050                                                            125 1.644 22 0.044                                                            126 1.409 23 0.043                                                            127 1.625 24 0.045                                                            128 1.061 25 0.046                                                            129 1.553 26 0.049                                                            130 1.709 27 0.050                                                            131 0.041 28 0.047                                                            132 0.044 29 0.050                                                            133 1.648 30 0.053                                                            134 0.043 31 0.051                                                            135 1.268 32 0.053                                                            136 1.480 33 0.055                                                            138 0.628 34 0.064                                                            139 0.042 35 0.063                                                            140 0.040 36 0.057                                                            141 0.039 38 0.048                                                            142 1.659 39 0.045                                                            143 1.457 40 0.046                                                            144 0.722 41 0.046                                                            145 1.256 42 0.051                                                            146 0.373 43 0.057                                                            147 1.732 44 0.050                                                            148 1.089 45 0.050                                                            149 1.606 46 0.045                                                            150 1.725 47 0.041                                                            151 1.449 48 0.064                                                            154 1.639 49 0.040                                                            155 1.775 50 0.036                                                          ______________________________________                                    

                  TABLE 6                                                         ______________________________________                                        Comparison of anti-HCV antibody detection by nylon-bound peptides,             a peptide-based ELISA, and a commercially available kit.                       Se-                                                                                   rum Nylon-bound peptides                                                      nr.  I   III IV  V   VI  VIII XI  XIV                                                    XV   XVI    XVIII                                        ______________________________________                                          191 0 0 0 0 0 0 0 0 0 0 0                                                     192 0 0 0 0 0 0 0 0 0 0 0                                                     193 0 0 0 0 0 0 0 0 0 0 0                                                     194 0 0 0 0 0 0 0 0 0 0 0                                                     195 1 2 2 3 0 0 0.5 0.5 1 3 1                                                 195 1 2 1 2 0.5 0.5 0.5 0.5 0.5 2 0                                           197 1 2 1 2 0 0.5 0.5 0.5 1 2 0                                               198 1 2 2 2 0 0 0 0 1 2 0                                                     211 0.5 1 0.5 0.5 0 2 2 0 2 0 1                                               213 0 0 0 1 0 0 0 0 0 0 0                                                     214 0 0 0 1 0 0 0 0 0 0 0                                                     215 0 0 0 1 0 0 0 0 0 0 0                                                     216 0 0 0 0.5 0 0 0 0 0 0 0                                                   217 0 0 0 1 0 0 0 0 0 0 0                                                     219 0.5 1 1 2 1 0.5 1 0 0.5 0.5 1                                             220 0.5 1 1 2 1 0.5 1 0 0.5 0.5 1                                             221 0 0 0 0.5 0 0 0 0 0 0 0                                                   222 1 1 1 1 0 0 2 0.5 0.5 0 0                                                 223 1 1 1 1 0 0 3 0.5 0.5 0 0                                                 224 1 1 2 1 0 0.5 3 0.5 0.5 0 0                                               225 0 0 0 0 0 0.5 0.5 0.5 0 0 2                                               226 0.5 0 0 0 0 2 3 2 0.5 0.5 3                                               227 0 0 0 0 0 2 2 0.5 0.5 0.5 2                                               228 0.5 0.5 0 0.5 0 2 2 2 0 0 2                                               234 0.5 0.5 0 0.5 0 0 3 1 3 1 3                                               235 0 0 0 0.5 0 0 0 0 0 0 0                                                   236 0 0 0 0.5 0 0 0 0 0 0 0                                                   237 0 0 0 1 0 0 0 0 0 0 0                                                     238 0 0 0 1 0 1 1 0 0 0 0                                                   ______________________________________                                               Optical density     Optical density                                      Serum nr. Peptide ELISA S/N Commercial ELISA S/N                            ______________________________________                                          191 0.045 0.18 0.295 0.47                                                     192 0.042 0.17 0.289 0.46                                                     193 0.039 0.16 0.197 0.32                                                     194 0.044 0.18 0.183 0.29                                                     195 1.692 6.77 3.000* 4.82*                                                   196 1.569 6.28 0.386 0.62                                                     197 1.523 6.09 0.447 0.72                                                     198 1.578 6.31 0.354 0.57                                                     211 1.606 6.42 3.000* 4.82*                                                   213 0.369 1.48 0.127 0.20                                                     214 0.444 1.78 0.101 0.16                                                     215 0.637 2.55 0.101 0.16                                                     216 0.812 3.25 0.092 0.15                                                     217 1.320 5.28 0.875 1.40                                                     219 1.547 6.19 3.000* 4.82*                                                   220 1.536 6.14 3.000* 4.82*                                                   221 1.428 5.71 0.327 0.52                                                     222 1.362 5.45 3.000* 4.82*                                                   223 1.316 5.26 3.000* 4.82*                                                   224 1.304 5.22 3.000* 4.82*                                                   225 1.178 4.71 2.398 3.85                                                     226 1.256 5.14 3.000* 4.82*                                                   227 1.335 5.34 3.000* 4.82*                                                   228 1.400 5.60 3.000* 4.82*                                                   234 1.481 5.92 3.000* 4.82*                                                   235 0.351 1.40 0.257 0.41                                                     236 0.475 1.90 0.245 0.39                                                     237 1.134 4.54 0.351 0.56                                                     238 1.096 4.38 1.074 1.72                                                   Cut-off: 0.250     Cut-off: 0.623                                             ______________________________________                                         0: no reaction; 0.5: weakly positive; 1: clearly positive; 2: strong          reaction; 3: intense reaction;                                                *O.D. exceeded 3.000 and was out of range. The values given are therefore     minimum values.                                                          

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 23                                          - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - - Met Ser Thr Ile Pro Lys Pro Gln Arg Lys Th - #r Lys Arg Asn Thr        Asn                                                                             1               5   - #                10  - #                15              - - Arg Arg Pro Gln                                                                      20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - - Pro Gln Arg Lys Thr Lys Arg Asn Thr Asn Ar - #g Arg Pro Gln Asp Val      1               5   - #                10  - #                15               - - Lys Phe Pro Gly                                                                      20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                               - - Gln Arg Lys Thr Lys Arg Asn Thr Asn Arg Ar - #g                          1               5   - #                10                                      - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                               - - Arg Asn Thr Asn Arg Arg Pro Gln Asp Val Ly - #s Phe Pro Gly Gly Gly      1               5   - #                10  - #                15               - - Gln Ile Val Gly                                                                      20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:5:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                               - - Leu Pro Arg Arg Gly Pro Arg Leu Gly Val Ar - #g Ala Thr Arg Lys Thr      1               5   - #                10  - #                15               - - Ser Glu Arg Ser                                                                      20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:6:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                               - - Thr Arg Lys Thr Ser Glu Arg Ser Gln Pro Ar - #g Gly Arg Arg Gln Pro      1               5   - #                10  - #                15               - - Ile Pro Lys Val                                                                      20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:7:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                               - - Arg Arg Gln Pro Ile Pro Lys Val Arg Arg Pr - #o Glu Gly Arg Thr Trp      1               5   - #                10  - #                15               - - Ala Gln Pro Gly                                                                      20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:8:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                               - - Gly Arg Thr Trp Ala Gln Pro Gly Tyr Pro Tr - #p Pro Leu Tyr Gly Asn      1               5   - #                10  - #                15               - - Glu Gly Cys Gly                                                                      20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:9:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                               - - Leu Ser Gly Lys Pro Ala Ile Ile Pro Asp Ar - #g Glu Val Leu Tyr Arg      1               5   - #                10  - #                15               - - Glu Phe Asp Glu                                                                      20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:10:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                              - - Ile Ile Pro Asp Arg Glu Val Leu Tyr Arg Gl - #u Phe Asp Glu Met Glu      1               5   - #                10  - #                15               - - Glu Cys Ser Gln                                                                      20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:11:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                              - - Asp Glu Met Glu Glu Cys Ser Gln His Leu Pr - #o Tyr Ile Glu Gln Gly      1               5   - #                10  - #                15               - - Met Met Leu Ala                                                                      20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:12:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                              - - Ser Gln His Leu Pro Tyr Ile Glu Gln Gly Me - #t Met Leu Ala Glu Gln      1               5   - #                10  - #                15               - - Phe Lys Gln Lys                                                                      20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:13:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                              - - Ile Glu Gln Gly Met Met Leu Ala Glu Gln Ph - #e Lys Gln Lys Ala Leu      1               5   - #                10  - #                15               - - Gly Leu Leu Gln                                                                      20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:14:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                              - - Leu Ala Glu Gln Phe Lys Gln Lys Ala Leu Gl - #y Leu Leu Gln Thr Ala      1               5   - #                10  - #                15               - - Ser Arg Gln Ala                                                                      20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:15:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                              - - Gln Lys Ala Leu Gly Leu Leu Gln Thr Ala Se - #r Arg Gln Ala Glu Val      1               5   - #                10  - #                15               - - Ile Ala Pro Ala                                                                      20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:16:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                              - - Glu Asp Glu Arg Glu Ile Ser Val Pro Ala Gl - #u Ile Leu Arg Lys Ser      1               5   - #                10  - #                15               - - Arg Arg Phe Ala                                                                      20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:17:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                              - - Leu Arg Lys Ser Arg Arg Phe Ala Gln Ala Le - #u Pro Val Trp Ala Arg      1               5   - #                10  - #                15               - - Pro Asp Tyr Asn                                                                      20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:18:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                              - - Val Trp Ala Arg Pro Asp Tyr Asn Pro Pro Le - #u Val Glu Thr Trp Lys      1               5   - #                10  - #                15               - - Lys Pro Asp Tyr                                                                      20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:19:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                              - - Glu Thr Trp Lys Lys Pro Asp Tyr Glu Pro Pr - #o Val Val His Gly Cys      1               5   - #                10  - #                15               - - Pro Leu Pro Pro                                                                      20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:20:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                              - - Val His Gly Cys Pro Leu Pro Pro Pro Lys Se - #r Pro Pro Val Pro Pro      1               5   - #                10  - #                15               - - Pro Arg Lys Lys                                                                      20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:21:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                              - - Glu Arg Glu Ile Ser Val Pro Ala Glu Ile Le - #u Arg Lys Ser Arg Arg      1               5   - #                10  - #                15               - -  - - (2) INFORMATION FOR SEQ ID NO:22:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                              - - Arg Phe Ala Gln Ala Leu Pro Val Trp Ala Ar - #g                          1               5   - #                10                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 23:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 2894 amino - #acids                                               (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #23:                          - - Met Ser Thr Ile Pro Lys Pro Gln Arg Lys Th - #r Lys Arg Asn Thr Asn      1               5   - #                10  - #                15               - - Arg Arg Pro Gln Asp Val Lys Phe Pro Gly Gl - #y Gly Gln Ile Val Gly                  20      - #            25      - #            30                   - - Gly Val Tyr Leu Leu Pro Arg Arg Gly Pro Ar - #g Leu Gly Val Arg Ala              35          - #        40          - #        45                       - - Thr Arg Lys Thr Ser Glu Arg Ser Gln Pro Ar - #g Gly Arg Arg Gln Pro          50              - #    55              - #    60                           - - Ile Pro Lys Val Arg Arg Pro Glu Gly Arg Th - #r Trp Ala Gln Pro Gly      65                  - #70                  - #75                  - #80        - - Tyr Pro Trp Pro Leu Tyr Gly Asn Glu Gly Cy - #s Gly Trp Ala Gly Trp                      85  - #                90  - #                95               - - Leu Leu Ser Pro Arg Gly Ser Arg Pro Ser Tr - #p Gly Pro Thr Asp Pro                  100      - #           105      - #           110                  - - Arg Arg Arg Ser Arg Asn Leu Gly Lys Val Il - #e Asp Thr Leu Thr Cys              115          - #       120          - #       125                      - - Gly Phe Ala Asp Leu Met Gly Tyr Ile Pro Le - #u Val Gly Ala Pro Leu          130              - #   135              - #   140                          - - Gly Gly Ala Ala Arg Ala Leu Ala His Gly Va - #l Arg Val Leu Glu Asp      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Gly Val Asn Tyr Ala Thr Gly Asn Leu Pro Gl - #y Cys Ser Phe Ser        Ile                                                                                             165  - #               170  - #               175             - - Phe Leu Leu Ala Leu Leu Ser Cys Leu Thr Va - #l Pro Ala Ser Ala Tyr                  180      - #           185      - #           190                  - - Gln Val Arg Asn Ser Thr Gly Leu Tyr His Va - #l Thr Asn Asp Cys Pro              195          - #       200          - #       205                      - - Asn Ser Ser Ile Val Tyr Glu Ala His Asp Al - #a Ile Leu His Thr Pro          210              - #   215              - #   220                          - - Gly Cys Val Pro Cys Val Arg Glu Gly Asn Va - #l Ser Arg Cys Trp Val      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Ala Met Thr Pro Thr Val Ala Thr Arg Asp Gl - #y Lys Leu Pro Ala        Thr                                                                                             245  - #               250  - #               255             - - Gln Leu Arg Arg His Ile Asp Leu Leu Val Gl - #y Ser Ala Thr Leu Cys                  260      - #           265      - #           270                  - - Ser Ala Leu Tyr Val Gly Asp Leu Cys Gly Se - #r Val Phe Leu Ile Gly              275          - #       280          - #       285                      - - Gln Leu Phe Thr Phe Ser Pro Arg Arg His Tr - #p Thr Thr Gln Gly Cys          290              - #   295              - #   300                          - - Asn Cys Ser Ile Tyr Pro Gly His Ile Thr Gl - #y His Arg Met Ala Trp      305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - Asp Met Met Met Asn Trp Ser Pro Thr Ala Al - #a Leu Val Met Ala        Gln                                                                                             325  - #               330  - #               335             - - Leu Leu Arg Ile Pro Gln Ala Ile Leu Asp Me - #t Ile Ala Gly Ala His                  340      - #           345      - #           350                  - - Trp Gly Val Leu Ala Gly Ile Ala Tyr Phe Se - #r Met Val Gly Asn Trp              355          - #       360          - #       365                      - - Ala Lys Val Leu Val Val Leu Leu Leu Phe Al - #a Gly Val Asp Ala Glu          370              - #   375              - #   380                          - - Thr Ile Val Ser Gly Gly Gln Ala Ala Arg Al - #a Met Ser Gly Leu Val      385                 3 - #90                 3 - #95                 4 -      #00                                                                              - - Ser Leu Phe Thr Pro Gly Ala Lys Gln Asn Il - #e Gln Leu Ile Asn        Thr                                                                                             405  - #               410  - #               415             - - Asn Gly Ser Trp His Ile Asn Ser Thr Ala Le - #u Asn Cys Asn Glu Ser                  420      - #           425      - #           430                  - - Leu Asn Thr Gly Trp Leu Ala Gly Leu Ile Ty - #r Gln His Lys Phe Asn              435          - #       440          - #       445                      - - Ser Ser Gly Cys Pro Glu Arg Leu Ala Ser Cy - #s Arg Pro Leu Thr Asp          450              - #   455              - #   460                          - - Phe Asp Gln Gly Trp Gly Pro Ile Ser Tyr Al - #a Asn Gly Ser Gly Pro      465                 4 - #70                 4 - #75                 4 -      #80                                                                              - - Asp Gln Arg Pro Tyr Cys Trp His Tyr Pro Pr - #o Lys Pro Cys Gly        Ile                                                                                             485  - #               490  - #               495             - - Val Pro Ala Lys Ser Val Cys Gly Pro Val Ty - #r Cys Phe Thr Pro Ser                  500      - #           505      - #           510                  - - Pro Val Val Val Gly Thr Thr Asp Arg Ser Gl - #y Ala Pro Thr Tyr Ser              515          - #       520          - #       525                      - - Trp Gly Glu Asn Asp Thr Asp Val Phe Val Le - #u Asn Asn Thr Arg Pro          530              - #   535              - #   540                          - - Pro Leu Gly Asn Trp Phe Gly Cys Thr Trp Me - #t Asn Ser Thr Gly Phe      545                 5 - #50                 5 - #55                 5 -      #60                                                                              - - Thr Lys Val Cys Gly Ala Pro Pro Cys Val Il - #e Gly Gly Ala Gly        Asn                                                                                             565  - #               570  - #               575             - - Asn Thr Leu His Cys Pro Thr Asp Cys Phe Ar - #g Lys His Pro Asp Ala                  580      - #           585      - #           590                  - - Thr Tyr Ser Arg Cys Gly Ser Gly Pro Trp Il - #e Thr Pro Arg Cys Leu              595          - #       600          - #       605                      - - Val Asp Tyr Pro Tyr Arg Leu Trp His Tyr Pr - #o Cys Thr Ile Asn Tyr          610              - #   615              - #   620                          - - Thr Ile Phe Lys Ile Arg Met Tyr Val Gly Gl - #y Val Glu His Arg Leu      625                 6 - #30                 6 - #35                 6 -      #40                                                                              - - Glu Ala Ala Cys Asn Trp Thr Arg Gly Glu Ar - #g Cys Asp Leu Glu        Asp                                                                                             645  - #               650  - #               655             - - Arg Asp Arg Ser Glu Leu Ser Pro Leu Leu Le - #u Thr Thr Thr Gln Trp                  660      - #           665      - #           670                  - - Gln Val Leu Pro Cys Ser Phe Thr Thr Leu Pr - #o Ala Leu Ser Thr Gly              675          - #       680          - #       685                      - - Leu Ile His Leu His Gln Asn Ile Val Asp Va - #l Gln Tyr Leu Tyr Gly          690              - #   695              - #   700                          - - Val Gly Ser Ser Ile Ala Ser Trp Ala Ile Ly - #s Trp Glu Tyr Val Val      705                 7 - #10                 7 - #15                 7 -      #20                                                                              - - Leu Leu Phe Leu Leu Leu Ala Asp Ala Arg Va - #l Cys Ser Cys Leu        Trp                                                                                             725  - #               730  - #               735             - - Met Met Leu Leu Ile Ser Gln Ala Glu Ala Al - #a Leu Glu Asn Leu Val                  740      - #           745      - #           750                  - - Ile Leu Asn Ala Ala Ser Leu Ala Gly Thr Hi - #s Gly Leu Val Ser Phe              755          - #       760          - #       765                      - - Leu Val Phe Phe Cys Phe Ala Trp Tyr Leu Ly - #s Gly Lys Trp Val Pro          770              - #   775              - #   780                          - - Gly Ala Val Tyr Thr Phe Tyr Gly Met Trp Pr - #o Leu Leu Leu Leu Leu      785                 7 - #90                 7 - #95                 8 -      #00                                                                              - - Leu Ala Leu Pro Gln Arg Ala Tyr Ala Leu As - #p Thr Glu Val Ala        Ala                                                                                             805  - #               810  - #               815             - - Ser Cys Gly Gly Val Val Leu Val Gly Leu Me - #t Ala Leu Thr Leu Ser                  820      - #           825      - #           830                  - - Pro Tyr Tyr Lys Arg Tyr Ile Ser Trp Cys Le - #u Trp Trp Leu Gln Tyr              835          - #       840          - #       845                      - - Phe Leu Thr Arg Val Glu Ala Gln Leu His Va - #l Trp Ile Pro Pro Leu          850              - #   855              - #   860                          - - Asn Val Arg Gly Gly Arg Asp Ala Val Ile Le - #u Leu Met Cys Ala Val      865                 8 - #70                 8 - #75                 8 -      #80                                                                              - - His Pro Thr Leu Val Phe Asp Ile Thr Lys Le - #u Leu Leu Ala Val        Phe                                                                                             885  - #               890  - #               895             - - Gly Pro Leu Trp Ile Leu Asp Ala Ser Leu Le - #u Lys Val Pro Tyr Phe                  900      - #           905      - #           910                  - - Val Arg Val Gln Gly Leu Leu Arg Phe Cys Al - #a Leu Ala Arg Lys Met              915          - #       920          - #       925                      - - Ile Gly Gly His Tyr Val Gln Met Val Ile Il - #e Lys Leu Gly Ala Leu          930              - #   935              - #   940                          - - Thr Gly Thr Tyr Val Tyr Asn His Leu Thr Pr - #o Leu Arg Asp Trp Ala      945                 9 - #50                 9 - #55                 9 -      #60                                                                              - - His Asn Gly Leu Arg Asp Leu Ala Val Ala Va - #l Glu Pro Val Val        Phe                                                                                             965  - #               970  - #               975             - - Ser Gln Met Glu Thr Lys Leu Ile Thr Trp Gl - #y Ala Asp Thr Ala Ala                  980      - #           985      - #           990                  - - Cys Gly Asp Ile Ile Asn Gly Leu Pro Val Se - #r Ala Arg Arg Gly Arg              995          - #       1000          - #      1005                     - - Glu Ile Leu Leu Gly Pro Ala Asp Gly Met Va - #l Ser Lys Gly Trp Arg          1010             - #   1015              - #  1020                         - - Leu Leu Ala Pro Ile Thr Ala Tyr Ala Gln Gl - #n Thr Arg Gly Leu Leu      1025                1030 - #                1035 - #               1040        - - Gly Cys Ile Ile Thr Ser Leu Thr Gly Arg As - #p Lys Asn Gln Val Glu                      1045 - #               1050  - #              1055             - - Gly Glu Val Gln Ile Val Ser Thr Ala Ala Gl - #n Thr Phe Leu Ala Thr                  1060     - #           1065      - #          1070                 - - Cys Ile Asn Gly Val Cys Trp Thr Val Tyr Hi - #s Gly Ala Gly Thr Arg              1075         - #       1080          - #      1085                     - - Thr Ile Ala Ser Pro Lys Gly Pro Val Ile Gl - #n Met Tyr Thr Asn Val          1090             - #   1095              - #  1100                         - - Asp Gln Asp Leu Val Gly Trp Pro Ala Pro Gl - #n Gly Ser Arg Ser Leu      1105                1110 - #                1115 - #               1120        - - Thr Pro Cys Thr Cys Gly Ser Ser Asp Leu Ty - #r Leu Val Thr Arg His                      1125 - #               1130  - #              1135             - - Ala Asp Val Ile Pro Val Arg Arg Arg Gly As - #p Ser Arg Gly Ser Leu                  1140     - #           1145      - #          1150                 - - Leu Ser Pro Arg Pro Ile Ser Tyr Leu Lys Gl - #y Ser Ser Gly Gly Pro              1155         - #       1160          - #      1165                     - - Leu Leu Cys Pro Ala Gly His Ala Val Gly Il - #e Phe Arg Ala Ala Val          1170             - #   1175              - #  1180                         - - Cys Thr Arg Gly Val Ala Lys Ala Val Asp Ph - #e Ile Pro Val Glu Asn      1185                1190 - #                1195 - #               1200        - - Leu Glu Thr Thr Met Arg Ser Pro Val Phe Tr - #p Asp Asn Ser Ser Pro                      1205 - #               1210  - #              1215             - - Pro Val Val Pro Gln Ser Phe Gln Val Ala Hi - #s Leu His Ala Pro Thr                  1220     - #           1225      - #          1230                 - - Gly Ser Gly Lys Ser Thr Lys Val Pro Ala Al - #a Tyr Ala Ala Gln Gly              1235         - #       1240          - #      1245                     - - Tyr Lys Val Leu Val Leu Asn Pro Ser Val Al - #a Ala Thr Leu Gly Phe          1250             - #   1255              - #  1260                         - - Gly Ala Tyr Met Ser Lys Ala His Gly Ile As - #p Pro Asn Ile Arg Thr      1265                1270 - #                1275 - #               1280        - - Gly Val Arg Thr Ile Thr Thr Gly Ser Pro Il - #e Thr Tyr Ser Thr Tyr                      1285 - #               1290  - #              1295             - - Gly Lys Phe Leu Ala Asp Gly Gly Cys Ser Gl - #y Gly Ala Tyr Asp Ile                  1300     - #           1305      - #          1310                 - - Ile Ile Cys Asp Glu Cys His Ser Thr Asp Al - #a Thr Ser Ile Leu Gly              1315         - #       1320          - #      1325                     - - Ile Gly Thr Val Leu Asp Gln Ala Glu Thr Al - #a Gly Ala Arg Leu Val          1330             - #   1335              - #  1340                         - - Val Leu Ala Thr Ala Thr Pro Pro Gly Ser Va - #l Thr Val Pro His Pro      1345                1350 - #                1355 - #               1360        - - Asn Ile Glu Glu Val Ala Leu Ser Thr Thr Gl - #y Glu Ile Pro Phe Tyr                      1365 - #               1370  - #              1375             - - Gly Lys Ala Ile Pro Leu Glu Val Ile Lys Gl - #y Gly Arg His Leu Ile                  1380     - #           1385      - #          1390                 - - Phe Cys His Ser Lys Lys Lys Cys Asp Glu Le - #u Ala Ala Lys Leu Val              1395         - #       1400          - #      1405                     - - Ala Leu Gly Ile Asn Ala Val Ala Tyr Tyr Ar - #g Gly Leu Asp Val Ser          1410             - #   1415              - #  1420                         - - Val Ile Pro Thr Ser Gly Asp Val Val Val Va - #l Ala Thr Asp Ala Leu      1425                1430 - #                1435 - #               1440        - - Met Thr Gly Tyr Thr Gly Asp Phe Asp Ser Va - #l Ile Asp Cys Asn Thr                      1445 - #               1450  - #              1455             - - Cys Val Thr Gln Thr Val Asp Phe Ser Leu As - #p Pro Thr Phe Thr Ile                  1460     - #           1465      - #          1470                 - - Glu Thr Ile Thr Leu Pro Gln Asp Ala Val Se - #r Arg Thr Gln Arg Arg              1475         - #       1480          - #      1485                     - - Gly Arg Thr Gly Arg Gly Lys Pro Gly Ile Ty - #r Arg Phe Val Ala Pro          1490             - #   1495              - #  1500                         - - Gly Glu Arg Pro Ser Gly Met Phe Asp Ser Se - #r Val Leu Cys Glu Cys      1505                1510 - #                1515 - #               1520        - - Tyr Asp Ala Gly Cys Ala Trp Tyr Glu Leu Th - #r Pro Ala Glu Thr Thr                      1525 - #               1530  - #              1535             - - Val Arg Leu Arg Ala Tyr Met Asn Thr Pro Gl - #y Leu Pro Val Cys Gln                  1540     - #           1545      - #          1550                 - - Asp His Leu Glu Phe Trp Glu Gly Val Phe Th - #r Gly Leu Thr His Ile              1555         - #       1560          - #      1565                     - - Asp Ala His Phe Leu Ser Gln Thr Lys Gly Se - #r Gly Glu Asn Leu Pro          1570             - #   1575              - #  1580                         - - Tyr Leu Val Ala Tyr Gln Ala Thr Val Cys Al - #a Arg Ala Gln Ala Pro      1585                1590 - #                1595 - #               1600        - - Pro Pro Ser Trp Asp Gln Met Trp Lys Cys Le - #u Ile Arg Leu Lys Pro                      1605 - #               1610  - #              1615             - - Thr Leu His Gly Pro Thr Pro Leu Leu Tyr Ar - #g Leu Gly Ala Val Gln                  1620     - #           1625      - #          1630                 - - Asn Glu Ile Thr Leu Thr His Pro Val Thr Ly - #s Tyr Ile Met Thr Cys              1635         - #       1640          - #      1645                     - - Met Ser Ala Asp Leu Glu Val Val Thr Ser Th - #r Trp Val Leu Val Gly          1650             - #   1655              - #  1660                         - - Gly Val Leu Ala Ala Leu Ala Ala Tyr Cys Le - #u Ser Thr Gly Cys Val      1665                1670 - #                1675 - #               1680        - - Val Ile Val Gly Arg Val Val Leu Ser Gly Ly - #s Pro Ala Ile Ile Pro                      1685 - #               1690  - #              1695             - - Asp Arg Glu Val Leu Tyr Arg Glu Phe Asp Gl - #u Met Glu Glu Cys Ser                  1700     - #           1705      - #          1710                 - - Gln His Leu Pro Tyr Ile Glu Gln Gly Met Me - #t Leu Ala Glu Gln Phe              1715         - #       1720          - #      1725                     - - Lys Gln Lys Ala Leu Gly Leu Leu Gln Thr Al - #a Ser Arg Gln Ala Glu          1730             - #   1735              - #  1740                         - - Val Ile Ala Pro Ala Val Gln Thr Asn Trp Gl - #n Lys Leu Glu Thr Phe      1745                1750 - #                1755 - #               1760        - - Trp Ala Lys His Met Trp Asn Phe Ile Ser Gl - #y Ile Gln Tyr Leu Ala                      1765 - #               1770  - #              1775             - - Gly Leu Ser Thr Leu Pro Gly Asn Pro Ala Il - #e Ala Ser Leu Met Ala                  1780     - #           1785      - #          1790                 - - Phe Thr Ala Ala Val Thr Ser Pro Leu Thr Th - #r Ser Gln Thr Leu Leu              1795         - #       1800          - #      1805                     - - Phe Asn Ile Leu Gly Gly Trp Val Ala Ala Gl - #n Leu Ala Ala Pro Gly          1810             - #   1815              - #  1820                         - - Ala Ala Thr Ala Phe Val Gly Ala Gly Leu Al - #a Gly Ala Ala Ile Gly      1825                1830 - #                1835 - #               1840        - - Ser Val Gly Leu Gly Lys Val Leu Ile Asp Il - #e Leu Ala Gly Tyr Gly                      1845 - #               1850  - #              1855             - - Ala Gly Val Ala Gly Ala Leu Val Ala Phe Ly - #s Ile Met Ser Gly Glu                  1860     - #           1865      - #          1870                 - - Val Pro Ser Thr Glu Asp Leu Val Asn Leu Le - #u Pro Ala Ile Leu Ser              1875         - #       1880          - #      1885                     - - Pro Gly Ala Leu Val Val Gly Val Val Cys Al - #a Ala Ile Leu Arg Arg          1890             - #   1895              - #  1900                         - - His Val Gly Pro Gly Glu Gly Ala Val Gln Tr - #p Met Asn Arg Leu Ile      1905                1910 - #                1915 - #               1920        - - Ala Phe Ala Ser Arg Gly Asn His Val Ser Pr - #o Thr His Tyr Val Pro                      1925 - #               1930  - #              1935             - - Glu Ser Asp Ala Ala Ala Arg Val Thr Ala Il - #e Leu Ser Ser Leu Thr                  1940     - #           1945      - #          1950                 - - Val Thr Gln Leu Leu Arg Arg Leu His Gln Tr - #p Ile Ser Ser Glu Cys              1955         - #       1960          - #      1965                     - - Thr Thr Pro Cys Ser Gly Ser Trp Leu Arg As - #p Ile Trp Asp Trp Ile          1970             - #   1975              - #  1980                         - - Cys Glu Val Leu Ser Asp Phe Lys Thr Trp Le - #u Lys Ala Lys Leu Met      1985                1990 - #                1995 - #               2000        - - Pro Gln Leu Pro Gly Ile Pro Phe Val Ser Cy - #s Gln Arg Gly Tyr Lys                      2005 - #               2010  - #              2015             - - Gly Val Trp Arg Val Asp Gly Ile Met His Th - #r Arg Cys His Cys Gly                  2020     - #           2025      - #          2030                 - - Ala Glu Ile Thr Gly His Val Lys Asn Gly Th - #r Met Arg Ile Val Gly              2035         - #       2040          - #      2045                     - - Pro Arg Thr Cys Arg Asn Met Trp Ser Gly Th - #r Phe Pro Ile Asn Ala          2050             - #   2055              - #  2060                         - - Tyr Thr Thr Gly Pro Cys Thr Arg Leu Pro Al - #a Pro Asn Tyr Thr Phe      2065                2070 - #                2075 - #               2080        - - Ala Leu Trp Arg Val Ser Ala Glu Glu Tyr Va - #l Glu Ile Arg Gln Val                      2085 - #               2090  - #              2095             - - Gly Asp Phe His Tyr Val Thr Gly Met Thr Th - #r Asp Asn Leu Lys Cys                  2100     - #           2105      - #          2110                 - - Pro Cys Gln Val Pro Ser Pro Glu Phe Phe Th - #r Glu Leu Asp Gly Val              2115         - #       2120          - #      2125                     - - Arg Leu His Arg Phe Ala Pro Pro Cys Lys Pr - #o Leu Leu Arg Glu Glu          2130             - #   2135              - #  2140                         - - Val Ser Phe Arg Val Gly Leu His Glu Tyr Pr - #o Val Gly Ser Gln Leu      2145                2150 - #                2155 - #               2160        - - Pro Cys Glu Pro Glu Pro Asp Val Ala Val Le - #u Thr Ser Met Leu Thr                      2165 - #               2170  - #              2175             - - Asp Pro Ser His Ile Thr Ala Glu Ala Ala Gl - #y Arg Arg Leu Ala Arg                  2180     - #           2185      - #          2190                 - - Gly Ser Pro Pro Ser Val Ala Ser Ser Ser Al - #a Ser Gln Leu Ser Ala              2195         - #       2200          - #      2205                     - - Pro Ser Leu Lys Ala Thr Cys Thr Ala Asn Hi - #s Asp Ser Pro Asp Ala          2210             - #   2215              - #  2220                         - - Glu Leu Ile Glu Ala Asn Leu Leu Trp Arg Gl - #n Glu Met Gly Gly Asn      2225                2230 - #                2235 - #               2240        - - Ile Thr Arg Val Glu Ser Glu Asn Lys Val Va - #l Ile Leu Asp Ser Phe                      2245 - #               2250  - #              2255             - - Asp Pro Leu Val Ala Glu Glu Asp Glu Arg Gl - #u Ile Ser Val Pro Ala                  2260     - #           2265      - #          2270                 - - Glu Ile Leu Arg Lys Ser Arg Arg Phe Ala Gl - #n Ala Leu Pro Val Trp              2275         - #       2280          - #      2285                     - - Ala Arg Pro Asp Tyr Asn Pro Pro Leu Val Gl - #u Thr Trp Lys Lys Pro          2290             - #   2295              - #  2300                         - - Asp Tyr Glu Pro Pro Val Val His Gly Cys Pr - #o Leu Pro Pro Pro Lys      2305                2310 - #                2315 - #               2320        - - Ser Pro Pro Val Pro Pro Pro Arg Lys Lys Ar - #g Thr Val Val Leu Thr                      2325 - #               2330  - #              2335             - - Glu Ser Thr Leu Ser Thr Ala Leu Ala Glu Le - #u Ala Thr Arg Ser Phe                  2340     - #           2345      - #          2350                 - - Gly Ser Ser Ser Thr Ser Gly Ile Thr Gly As - #p Asn Thr Thr Thr Ser              2355         - #       2360          - #      2365                     - - Ser Glu Pro Ala Pro Ser Gly Cys Pro Pro As - #p Ser Asp Ala Glu Ser          2370             - #   2375              - #  2380                         - - Tyr Ser Ser Met Pro Pro Leu Glu Gly Glu Pr - #o Gly Asp Pro Asp Leu      2385                2390 - #                2395 - #               2400        - - Ser Asp Gly Ser Trp Ser Thr Val Ser Ser Gl - #u Ala Asn Ala Glu Asp                      2405 - #               2410  - #              2415             - - Val Val Cys Cys Ser Met Ser Tyr Ser Trp Th - #r Gly Ala Cys Val Thr                  2420     - #           2425      - #          2430                 - - Pro Cys Ala Ala Glu Glu Gln Lys Leu Pro Il - #e Asn Ala Leu Ser Asn              2435         - #       2440          - #      2445                     - - Ser Leu Leu Arg His His Asn Leu Val Tyr Se - #r Thr Thr Ser Arg Ser          2450             - #   2455              - #  2460                         - - Ala Cys Gln Arg Gln Lys Lys Val Thr Phe As - #p Arg Leu Gln Val Leu      2465                2470 - #                2475 - #               2480        - - Asp Ser His Tyr Gln Asp Val Leu Lys Glu Va - #l Lys Ala Ala Ala Ser                      2485 - #               2490  - #              2495             - - Lys Val Lys Ala Asn Leu Leu Ser Val Glu Gl - #u Ala Cys Ser Leu Thr                  2500     - #           2505      - #          2510                 - - Pro Pro His Ser Ala Lys Ser Lys Phe Gly Ty - #r Gly Ala Lys Asp Val              2515         - #       2520          - #      2525                     - - Arg Cys His Ala Arg Lys Ala Val Thr His Il - #e Asn Ser Val Trp Lys          2530             - #   2535              - #  2540                         - - Asp Leu Leu Glu Asp Asn Val Thr Pro Ile As - #p Thr Thr Ile Met Ala      2545                2550 - #                2555 - #               2560        - - Lys Asn Glu Val Phe Cys Val Gln Pro Glu Ly - #s Gly Gly Arg Lys Pro                      2565 - #               2570  - #              2575             - - Ala Arg Leu Ile Val Phe Pro Asp Leu Gly Va - #l Arg Val Cys Glu Lys                  2580     - #           2585      - #          2590                 - - Met Ala Leu Tyr Asp Val Val Thr Lys Leu Pr - #o Leu Ala Val Met Gly              2595         - #       2600          - #      2605                     - - Ser Ser Tyr Gly Phe Gln Tyr Ser Pro Gly Gl - #n Arg Val Glu Phe Leu          2610             - #   2615              - #  2620                         - - Val Gln Ala Trp Lys Ser Lys Lys Thr Pro Me - #t Gly Phe Ser Tyr Asp      2625                2630 - #                2635 - #               2640        - - Thr Arg Cys Phe Asp Ser Thr Val Thr Glu Se - #r Asp Ile Arg Thr Glu                      2645 - #               2650  - #              2655             - - Glu Ala Ile Tyr Gln Cys Cys Asp Leu Asp Pr - #o Gln Ala Arg Val Ala                  2660     - #           2665      - #          2670                 - - Ile Lys Ser Leu Thr Glu Arg Leu Tyr Val Gl - #y Gly Pro Leu Thr Asn              2675         - #       2680          - #      2685                     - - Ser Arg Gly Glu Asn Cys Gly Tyr Arg Arg Cy - #s Arg Ala Ser Gly Val          2690             - #   2695              - #  2700                         - - Leu Thr Thr Ser Cys Gly Asn Thr Leu Thr Cy - #s Tyr Ile Lys Ala Arg      2705                2710 - #                2715 - #               2720        - - Ala Ala Cys Arg Ala Ala Gly Leu Gln Asp Cy - #s Thr Met Leu Val Cys                      2725 - #               2730  - #              2735             - - Gly Asp Asp Leu Val Val Ile Cys Glu Ser Al - #a Gly Val Gln Glu Asp                  2740     - #           2745      - #          2750                 - - Ala Ala Ser Leu Arg Ala Phe Thr Glu Ala Me - #t Thr Arg Tyr Ser Ala              2755         - #       2760          - #      2765                     - - Pro Pro Gly Asp Pro Pro Gln Pro Glu Tyr As - #p Leu Glu Leu Ile Thr          2770             - #   2775              - #  2780                         - - Ser Cys Ser Ser Asn Val Ser Val Ala His As - #p Gly Ala Gly Lys Arg      2785                2790 - #                2795 - #               2800        - - Val Tyr Tyr Leu Thr Arg Asp Pro Thr Thr Pr - #o Leu Ala Arg Ala Ala                      2805 - #               2810  - #              2815             - - Trp Glu Thr Ala Arg His Thr Pro Val Asn Se - #r Trp Leu Gly Asn Ile                  2820     - #           2825      - #          2830                 - - Ile Met Phe Ala Pro Thr Leu Trp Ala Arg Me - #t Ile Leu Met Thr His              2835         - #       2840          - #      2845                     - - Phe Phe Ser Val Leu Ile Ala Arg Asp Gln Le - #u Glu Gln Ala Leu Asp          2850             - #   2855              - #  2860                         - - Cys Glu Ile Tyr Gly Ala Cys Tyr Ser Ile Gl - #u Pro Leu Asp Leu Pro      2865                2870 - #                2875 - #               2880        - - Pro Ile Ile Gln Arg Leu Gly Cys Pro Glu Ar - #g Leu Ala Ser                              2885 - #               2890                                  __________________________________________________________________________

We claim:
 1. An isolated peptide having the amino acid sequence shown inSEQ ID NO:1.
 2. An isolated peptide having the amino acid sequence shownin SEQ ID NO:2.
 3. An isolated peptide having the amino acid sequenceshown in SEQ ID NO:4.
 4. An isolated peptide having the amino acidsequence shown in SEQ ID NO:5.
 5. An isolated peptide having the aminoacid sequence shown in SEQ ID NO:6.
 6. An isolated peptide having theamino acid sequence shown in SEQ ID NO:7.
 7. An isolated peptide havingthe amino acid sequence shown in SEQ ID NO:8.
 8. The peptide accordingto any one of claims 1-7, wherein said peptide is coupled N-terminally,C-terminally or internally to a carrier molecule.
 9. The peptideaccording to any one of claims 1-7, wherein said peptide contains adetectable label.
 10. The peptide of any one of claims 1-7, said peptidebeing cyclic.
 11. A method for the detection of antibodies to hepatitisC virus (HCV) present in a body fluid comprising the steps of:(a)contacting the body fluid with the peptide according to claim 10, and(b) detecting an immunological complex formed between antibodies to HCVin said body fluid and said peptide, the presence of said complex beingindicative of the presence of antibodies to HCV in said body fluid. 12.A kit for the detection of anti-hepatitis C virus antibodies in a bodyfluid, comprising:the peptide according to claim 10, and a means fordetecting an immunological complex formed between said peptide and saidantibodies.
 13. The peptide as in any one of claims 1-7, wherein saidpeptide is cyclic and is immunoreactive with HCV antibodies.
 14. Thepeptide as in any one of claims 1-7, wherein said peptide has on itsamino terminus an H, or one or more chemical linking groups, and has onits carboxy terminus an NH₂, or one or more chemical linking groups. 15.A method for the detection of antibodies to hepatitis C virus (HCV)present in a body fluid comprising the steps of:(a) contacting the bodyfluid with the peptide according to any one of claims 1-7, and (b)detecting an immunological complex formed between antibodies to HCV insaid body fluid and said peptide, the presence of said complex beingindicative of the presence of antibodies to HCV in said body fluid. 16.The method of claim 15, where said peptide is present as lines on anylon membrane.
 17. The method of claim 16, wherein said nylon membraneis cut into strips perpendicular to the direction of the peptide lines,and said strip is incubated with diluted serum sample.
 18. The method ofclaim 15, wherein said peptide is present in wells of microtiter plates.19. A kit for the detection of anti-hepatitis C virus antibodies in abody fluid, comprising:the peptide according to any one of claims 1-7,and a means for detecting an immunological complex formed between saidpeptide and said antibodies.
 20. The kit of claim 19, further comprisinga nylon membrane, said peptide being present as lines on said membrane,said membrane being cut into strips perpendicular to the direction ofthe peptide lines, such that each strip can be incubated with a dilutedserum sample.
 21. The kit of claim 19, further comprising a microtiterplate, said peptide being present in the wells of said microtiter plate.22. A peptide composition comprising the peptide of any one of claims1-7 and at least one additional peptide selected from the groupconsisting of peptides having amino acid sequences shown in SEQ ID NO:9,SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14,SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19,SEQ ID NO:20.
 23. The peptide composition according to claim 22, whereinat least one peptide is coupled N-terminally, C-terminally or internallyto a carrier molecule.
 24. The peptide composition according to claim22, wherein said composition contains a detectable label.
 25. Thepeptide composition as in claim 22, wherein at least one peptide iscyclic and is immunoreactive with HCV antibodies.
 26. The peptidecomposition as in claim 22, wherein at least one peptide has on itsamino terminus an H, or one or more chemical linking groups, and has onits carboxy terminus an NH₂, or one or more chemical linking groups. 27.A kit for the detection of anti-hepatitis C virus antibodies in a bodyfluid, comprising:the peptide composition according to claim 22, and ameans for detecting an immunological complex formed between said peptidecomposition and said antibodies.
 28. A method for the detection ofantibodies to hepatitis C virus (HCV) present in a body fluid comprisingthe steps of:(a) contacting the body fluid with the peptide compositionaccording to claim 22, and (b) detecting an immunological complex formedbetween antibodies to HCV in said body fluid and said peptidecomposition, the presence of said complex being indicative of thepresence of antibodies to HCV in said body fluid.
 29. The method ofclaim 28, where said peptides of said composition are present as lineson a nylon membrane.
 30. The method of claim 29, wherein said nylonmembrane is cut into strips perpendicular to the direction of thepeptide composition lines, and each strip is incubated with dilutedserum sample.
 31. The method of claim 30, wherein said peptidecomposition is present in wells of microtiter plates.
 32. The peptidecomposition of claim 22, wherein two or more peptides are joinedtogether, the joined peptide being capable of providing immunologicalcompetition with at least one strain of HCV.
 33. A kit for the detectionof anti-hepatitis C virus antibodies in a body fluid, comprising:thepeptide composition according to claim 32, and a means for detecting animmunological complex formed between said peptide composition and saidantibodies.
 34. A method for the detection of antibodies to hepatitis Cvirus (HCV) present in a body fluid comprising the steps of:(a)contacting the body fluid with the peptide composition according toclaim 32, and (b) detecting an immunological complex formed betweenantibodies to HCV in said body fluid and said peptide composition, thepresence of said complex being indicative of the presence of antibodiesto HCV in said body fluid.
 35. The peptide composition of claim 22,wherein at least one peptide is cyclic.
 36. A kit for the detection ofanti-hepatitis C virus antibodies in a body fluid, comprising:thepeptide composition according to claim 35, and a means for detecting animmunological complex formed between said peptide composition and saidantibodies.
 37. A method for the detection of antibodies to hepatitis Cvirus (HCV) present in a body fluid comprising the steps of:(a)contacting the body fluid with the peptide composition according toclaim 35, and (b) detecting an immunological complex formed betweenantibodies to HCV in said body fluid and said peptide composition, thepresence of said complex being indicative of the presence of antibodiesto HCV in said body fluid.
 38. A peptide consisting of two or morepeptides joined together, said two or more peptides being selected fromthe group consisting of peptides having amino acid sequences shown inSEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ IDNO:7, and SEQ ID NO:8,wherein said two or more peptides joined togetheris capable of providing immunological competition with at least onestrain of HCV.